CIRCULATING AUTOANTIBODIES RECOGNIZING PEROXIDASE-OXIDIZED LOW-DENSITY-LIPOPROTEIN - EVIDENCE FOR NEW ANTIGENIC EPITOPES FORMED IN-VIVO INDEPENDENTLY FROM LIPID-PEROXIDATION

Oxidatively modified LDLs are antigenic and elicit the generation of a utoantibodies often detected in plasma and within plaques of atheroscl erotic patients. Although Cu2+-oxidized LDL and malondialdehyde (MDA)- modified LDL are usually used as antigens in immunoassays, other, stil l unrecognized epitopes may be formed in vivo during LDL oxidation and may induce antibody production. Antibodies recognizing LDL oxidativel y modified by Cu2+, 2,2'-azobis-(2-amidino propane) hydrochloride (AAP H), and the combination of horseradish peroxidase and H2O2 (HRP) were detected in serum of a group of 90 unselected patients. HRP-oxidized L DL was the antigen that revealed the highest IgG titers, although the extent of LDL oxidation (evaluated as conjugated diene formation, loss of tryptophan fluorescence, production of fluorescent aldehydic adduc ts, and change in electrophoretic mobility) was comparable to that obt ained with Cu2+ and AAPH. There was a highly statistically significant correlation between the IgG titers detected using Cu2+- and AAPH-oxid ized LDLs as antigens, but no correlation was found between the Ige ti ters revealed by HRP and Cu2+ or AAPH. Ln addition, the antibody titer s against MDA-modified LDL exhibited a significant correlation with th ose against Cu2+- or AAPH-oxidized LDL but did not correlate with thos e against HRP-oxidized LDL. Finally, immunocompetition experiments rev ealed that IgG recognizing HRP-oxidized LDL did not crossreact with Cu 2+-oxidized LDL and vice versa. The possibility that lipid peroxidatio n-independent modifications could play a role in HRP-induced formation of antigenic epitopes in LDL was supported by two lines of evidence. First, in probucol-enriched LDL, despite the complete inhibition of li pid peroxidation, HRP, but not Cu2+ and AAPH, was still able to genera te epitopes that were recognized by the same sera reacting with HRP-ox idized native (not probucol-enriched) LDL. In addition, the presence o f autoantibodies against Cu2+- and AAPH-oxidized LDLs was negatively c orrelated with serum alpha-tocopherol concentration, whereas the titer s against HRP-oxidized LDL did not exhibit any statistically relevant correlation with cu-tocopherol levels. Together, these findings indica te that peroxidase(s)dependent mechanisms can trigger peculiar lipid p eroxidation-independent modifications of LDL in vivo.